Human stanniocalcin-alpha

ABSTRACT

A human stanniocalcin-alpha polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for the regulation of electrolyte imbalances which can lead to renal, bone and heart diseases and osteoporosis and Paget&#39;s Disease. Antagonists against such polypeptides and their use in the regulation of electrolyte imbalances which can lead to hypocalcemia and osteoporosis are also disclosed. Use of the stanniocalcin-alpha sequence as a diagnostic to detect diseases or the susceptibility to diseases related to a mutated form of stanniocalcin-alpha sequences is also disclosed.

This application is a Divisional of U.S. application Ser. No. 10/418,226filed Apr. 18, 2003 (allowed), which in turn is a Divisional of U.S.application Ser. No. 09/361,736 filed Jul. 28, 1999 (now U.S. Pat. No.6,613,877, issued Sep. 2, 2003), which in turn is a Divisional of U.S.application Ser. No. 08/460,529 filed Jun. 2, 1995 (now U.S. Pat. No.5,994,103, issued Nov. 30, 1999), which in turn is aContinuation-In-Part of International Application No. PCT/US94/13206filed Nov. 10, 1994. Each of the above-listed applications is herebyincorporated by reference in its entirety.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention has putatively been identified as human stanniocalcin-alpha.The invention also relates to inhibiting the action of suchpolypeptides.

Stanniocalcin (formerly known as both teleocalcin and hypocalcin) is ananti-hypercalcemic, glycoprotein hormone that is produced by thecorpuscles of stannius, endocrine glands of the bony fishes. Humans alsoproduce a stanniocalcin glycoprotein.

Stanniocalcin-alpha has similar reported biological activities toparathyroid hormone (PTH) and both of these proteins exhibit dualfunctions in mammals. They exert hypercalcemic activity possibly due tostimulation of bone resorption (Endocrinology 119:2249-2255 (1986)) andhypocalcaemic activity in fish. The hypocalcaemic activity is possiblydue to inhibition of gill calcium influx (J. Exp. Biol., 140:199-208(1988)). Further, PTH has a biphasic action on bone metabolism, i.e., atlow doses it increases bone formation, while at high doses it increasesbone resorption. Accordingly, both the polypeptide itself and anantagonist, under different circumstances, may be used to treatosteoporosis.

The Corpuscles of Stannius protein of non-humans has been studiedextensively. Recently, a Corpuscles of Stannius protein has beenpurified and cloned from Anguilla australis. The kidneys of teleost fishhave been found to contain secretory granules, the Corpuscles ofStannius. Electron microscopy indicates that the granules are of aproteinaceous nature and may represent hormones or enzymes ofunrecognized physiological and biochemical function (Butkus, A. et al.Mol. Cell Endocrinol, 54:123-33 (1987)).

There has also been isolated and purified a glycoprotein from theCorpuscles of Stannius of trout, which is considered hypocalcin, themajor hypocalcemic hormone of fish. This product is present inrelatively large amounts in the Corpuscles of Stannius of severalspecies (i.e., European eel, tilapia goldfish, and carp). Hypocalcin istypically released from the Corpuscles of Stannius in response to anexperimentally induced increase of the blood calcium concentration.Ultrastructural observations show that after this treatment thehypocalcin-producing cell type of the corpuscles of stannius are almostcompletely degranulated. The isolated glycoprotein has an apparentmolecular weight of 54 kDa. (Lafeber F. P. et al., Gen Comp. Endocrinol,69:19-30 (1988)).

Moreover, it has recently been shown that several synthetic peptidefragments of teleocalcin inhibit calcium uptake in juvenile rainbowtrout (Salmo Gairdneri). The N-terminal peptides (amino acids 1 to 20)of both eel and salmon teleocalcin significantly inhibit ⁴⁵Ca uptake atthe high point of the calcium uptake cycle (up to 75%), although theeffective doses of the peptides on a molar basis were 20 to 200 timesthat of the intact molecule. In contrast, the C-terminal fragment of eelteleocalcin (amino acids 202 to 231) did not have an inhibitory effecton calcium uptake (Milliken C. E. et al., Gen. Comp. Endocrinol,77:416-22 (1990)).

There has also been a description of the purification andcharacterization of two salmon stanniocalcins, and the examination ofthe regulation of hormone secretion in response to calcium using both invitro and in vivo model systems. The molecular cloning and cDNA sequenceanalysis of a Coho salmon stanniocalcin messenger RNA (mRNA) from asalmon CS lambda gt10 cDNA library is described. The stanniocalcin mRNAin salmon is approximately 2 kDa in length and encodes a primarytranslation product of 256 amino acids. The first 33 residues comprisethe preprotein region of the hormone, whereas the remaining 223 residuesmake up the mature form of the hormone. (Wagner G. F. et al., Mol. CellEndocrinol, 90:7-15 (1992)).

The polypeptide of the present invention has been putatively identifiedas human stanniocalcin-alpha. This identification has been made as aresult of amino acid sequence homology.

In accordance with one aspect of the present invention, there isprovided a novel putative mature polypeptide which is humanstanniocalcin-alpha, as well as biologically active and diagnosticallyor therapeutically useful fragments, analogs and derivatives thereof.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding humanstanniocalcin-alpha, including mRNAs, DNAs, cDNAs, genomic DNA as wellas antisense analogs thereof and biologically active and diagnosticallyor therapeutically useful fragments thereof.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a human stanniocalcin-alpha nucleicacid sequence, under conditions promoting expression of said protein andsubsequent recovery of said protein.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptide, or polynucleotideencoding such polypeptide, for therapeutic purposes, for example, totreat electrolyte disorders which lead to renal, and heart diseases and,due to a biphasic action of the polypeptide it may be employed to treat,osteoporosis, Paget's Disease and osteopetrosis.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, in the treatmentof osteoporosis and hypocalcemia. Hypocalcemia can arise from a numberof different causes including renal failure, hyperparathyroidism, severeinfections, pancreatic insufficiency or bums which trap calcium from theinter-cellular fluid. Hypocalcemia results in tetany, convulsions andother related disorders.

In accordance with still another aspect of the present invention, thereare provided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to human stanniocalcin-alphasequences.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIGS. 1A-B display the cDNA sequence (SEQ ID NO: 1) and correspondingdeduced amino acid sequence (SEQ ID NO:2) of the humanstanniocalcin-alpha protein. The standard three-letter abbreviations foramino acids are used.

FIG. 2 is a comparison of portions of the amino acid sequences ofstanniocalcin from Anguilla australis (lower line, SEQ ID NO:9) and fromhuman stanniocalcin-alpha (upper line, SEQ ID NO:2, from amino acid 39to amino acid 209). In the comparison, one-letter codes are used torepresent the amino acid residues of the amino acid sequences. From theFIG. 2 comparison, 35% of the amino acid residues are identical in a 170amino acid overlap to provide a total similarity of 55%.

FIG. 3 is a comparison of the amino acid sequences of humanstanniocalcin-alpha according to the invention (lower line, SEQ ID NO:2)and from human stanniocalcin (upper line, SEQ ID NO:10). In thecomparison, one-letter codes are used to represent the amino acidresidues of the amino acid sequences.

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIGS. 1A-B (SEQ IDNO:2) or for the mature polypeptide encoded by the cDNA of the clonedeposited as ATCC™ Deposit No. 75831 on Jul. 15, 1994 (SEQ ID NO: 12).

The ATCC™ number referred to above is directed to a biological depositwith the ATCC™, 10801 University Boulevard, Manassas, Va. 20110-2209.Since the strain referred to is being maintained under the terms of theBudapest Treaty, it will be made available to a patent office signatoryto the Budapest Treaty.

The polynucleotide of this invention was discovered in a cDNA libraryderived from lung fibroblast cells. It is structurally related to thehuman stanniocalcin family. It contains an open reading frame encoding aprotein of about 251 amino acid residues of which approximately thefirst 40 amino acids residues are the putative leader sequence such thatthe mature protein comprises 211 amino acids. The protein exhibits thehighest degree of homology to human stanniocalcin with 28% identity and64% similarity over the entire amino acid stretch.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIGS. 1A-B or that of the deposited cloneor may be a different coding sequence which coding sequence, as a resultof the redundancy or degeneracy of the genetic code, encodes the samemature polypeptide as the DNA of FIGS. 1A-B (SEQ ID NO: 1) or thedeposited cDNA (SEQ ID NO: 11).

The polynucleotide which encodes for the mature polypeptide of FIGS.1A-B or for the mature polypeptide encoded by the deposited cDNA (SEQ IDNO:12) may include: only the coding sequence for the mature polypeptide;the coding sequence for the mature polypeptide and additional codingsequence such as a leader or secretory sequence or a proproteinsequence; the coding sequence for the mature polypeptide (and optionallyadditional coding sequence) and non-coding sequence, such as introns ornon-coding sequence 5′ and/or 3′ of the coding sequence for the maturepolypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIGS. 1A-B or the polypeptide encoded by the cDNA of the deposited clone(SEQ ID NO:12). The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIGS. 1A-B or the same mature polypeptideencoded by the cDNA of the deposited clone (SEQ ID NO:12) as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIGS. 1A-B or the polypeptideencoded by the cDNA of the deposited clone. Such nucleotide variantsinclude deletion variants, substitution variants and addition orinsertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIGS. 1A-B or of the coding sequence of the deposited clone. Asknown in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains. Thus,for example, the polynucleotide of the present invention may encode fora mature protein, or for a protein having a prosequence or for a proteinhaving both a prosequence and a presequence (leader sequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence is preferably a hexa-histidine tag supplied by a vector, forexample a pQE-9 or pQE-60 vector, to provide for purification of themature polypeptide fused to the marker in the case of a bacterial host,or, for example, the marker sequence may be a hemagglutinin (HA) tagwhen a mammalian host, e.g. COS-7 cells, is used. The HA tag correspondsto an epitope derived from the influenza hemagglutinin protein (Wilson,I., et al., Cell, 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full-length polynucleotide of the invention may be usedas a hybridization probe for a cDNA library to isolate the full-lengthcDNA and to isolate other cDNA which have a high sequence similarity tothe full-length polynucleotide of the invention. Probes of this typepreferably have at least 10, 20 or 30 bases and may contain, forexample, 50 or more bases. The probe may also be used to identify a cDNAclone corresponding to a full length transcript and a genomic clone orclones that contain the complete gene of the invention includingregulatory and promotor regions, exons, and introns. An example of ascreen comprises isolating the coding region of the full-length gene ofthe invention by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 50% andpreferably 70% identity between the sequences. The present inventionparticularly relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides. As herein used,the term “stringent conditions” means hybridization will occur only ifthere is at least 95% and preferably at least 97% identity between thesequences. The polynucleotides which hybridize to the hereinabovedescribed polynucleotides in a preferred embodiment encode polypeptideswhich retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNA of FIGS. 1A-B or thedeposited cDNA (SEQ ID NO: 11).

Alternatively, the polynucleotide may have at least 20 bases, preferablyat least 30 bases, and more preferably at least 50 bases which hybridizeto a polynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

The present invention further relates to a human stanniocalcin-alphapolypeptide which has the deduced amino acid sequence of FIGS. 1A-B orwhich has the amino acid sequence encoded by the deposited cDNA (SEQ IDNO:12), as well as fragments, analogs and derivatives of suchpolypeptide.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide of FIGS. 1A-B or that encoded by the deposited cDNA (SEQ IDNO:12), means a polypeptide which retains essentially the samebiological function or activity as such polypeptide. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the mature polypeptide of FIGS.1A-B (SEQ ID NO:2) or that encoded by the deposited cDNA (SEQ ID NO:12)may be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues include asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct MRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), Â-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC™ 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The polypeptide can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

Human stanniocalcin-alpha administration may be used for therapeutictreatment of numerous electrolyte-based diseases. One cause of arterialhypertension is abnormal Na⁺ transport across the cell wall of thevascular smooth muscle cells due to a defect in or inhibition of theNa⁺-K+ pump, another is increased permeability to Na⁺ as has beendescribed in some forms of human hypertension. The net result is anincrease in intra-cellular Na⁺, which makes the cell more sensitive tovasoconstrictive agents. Since Ca⁺⁺ follows Na⁺, it is postulated thatit is the accumulation of intra-cellular Ca⁺⁺ and not Na⁺ per se that isresponsible for increased sensitivity to sympathetic stimulation.Accordingly, since human stanniocalcin-alpha can function as ahypocalcemic agent, it can help to offset this increased intra-cellularCa⁺⁺ and reduce or prevent hypertension.

Further, hypercalcemia has been implicated in heart dysrhythmias, comaand cardiac arrest. Accordingly, human stanniocalcin-alpha may havetherapeutic value for the treatment of these disorders by lowering theconcentration of free Ca⁺⁺.

Hypertension is also directly related to renal disorders. Accordingly, ahigher or lower than normal concentration of electrolytes can causerenal malfunction and directly lead to other disorders. As an example,Ca⁺⁺-phosphorous imbalance can cause muscle and bone pain,demineralization of the bones and calcification in various organsincluding the brain, eyes, myocardia and blood vessels. Accordingly, thepolypeptide of the present invention may be used to offset disordersthat are due to a Ca⁺⁺-phosphate imbalance. Renal failure itself leadsto an abnormally high concentration of phosphate in the blood which canbe reduced to normal concentrations by human stanniocalcin-alpha.

Human stanniocalcin-alpha is also useful for the treatment of certainbone diseases, in that, it may have a biphasic action on bonemetabolism, i.e., at low doses it may increase bone formation, while athigh doses it increases bone resorption. Therefore, administration oflow doses of human stanniocalcin-alpha may be employed to treatosteoporosis and administration of high doses may be employed to treatosteopetrosis, which is an overgrowth and sclerosis of bone with themarked thickening of the bony cortex and narrowing or filling of themarrow cavity.

The causes of hypercalcemia may also be a number of different disordersincluding hyperparathyroidism, hypervitaminosis D, tumors that raise theserum Ca⁺ levels by destroying bone, sarcoidosis, hyperthyroidism,adrenal insufficiency, loss of serum albumin, secondary renal diseases,excessive gastrointestinal calcium absorption and elevated concentrationof plasma proteins. Accordingly, human stanniocalcin-alpha is effectivein reducing hypercalcemia and its related disorders.

Human stanniocalcin-alpha may also be employed for the treatment ofother disorders relating to unusual electrolyte concentrations and fluidimbalance, for example, migraine headaches.

This invention provides a method for identification of humanstanniocalcin-alpha receptors. The gene encoding the receptor can beidentified by numerous methods known to those of skill in the art, forexample, ligand panning and FACS sorting (Coligan, et al., CurrentProtocols in Immun., 1(2), Chapter 5, (1991)). Preferably, expressioncloning is employed wherein polyadenylated RNA is prepared from a cellresponsive to human stanniocalcin-alpha, and a cDNA library created fromthis RNA is divided into pools and used to transfect COS cells or othercells that are not responsive to the human stanniocalcin-alpha protein.Transfected cells which are grown on glass slides are exposed to labeledhuman stanniocalcin-alpha. The stanniocalcin-alpha can be labeled by avariety of means including iodination or inclusion of a recognition sitefor a site-specific protein kinase. Following fixation and incubation,the slides are subjected to autoradiographic analysis. Positive poolsare identified and sub-pools are prepared and retransfected using aniterative sub-pooling and rescreening process, eventually yielding asingle clone that encodes the putative receptor. As an alternativeapproach for receptor identification, labeled human stanniocalcin-alphacan be photoaffinity linked with cell membrane or extract preparationsthat express the receptor molecule. Cross-linked material is resolved byPAGE and exposed to X-ray film. The labeled complex containing theligand-receptor can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing would be used to design a set of degenerateoligonucleotide probes to screen a cDNA library to identify the geneencoding the putative receptor.

This invention also provides a method of screening compounds to identifyagonists and antagonists of human stanniocalcin-alpha. As an example, abioassay may be performed wherein the assay components comprise amammalian cell or membrane preparation expressing a humanstanniocalcin-alpha receptor on the surface thereof, labeled calcium,for example ⁴⁵Ca⁺⁺, and the compound to be screened. If the compound isan effective human stanniocalcin-alpha agonist it will mimic the humanstanniocalcin-alpha receptor ligand such that there is ⁴⁵Ca⁺⁺ uptake bythe cell or membrane in the absence of human stanniocalcin-alpha. Theamount of ⁴⁵Ca⁺⁺ uptake can be determined by taking advantage of theradioactive label. When screening for an antagonist, humanstanniocalcin-alpha is added to the bioassay and the ability of thecompound to inhibit ⁴⁵Ca⁺⁺ uptake by interfering with the interaction ofhuman stanniocalcin-alpha and its receptor can be determined in the samemanner.

Alternatively, the response of a known second messenger system followinginteraction of human stanniocalcin-alpha and the receptor would bemeasured and compared in the presence and absence of the compound. Suchsecond messenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis.

Potential human stanniocalcin-alpha antagonists include antibodies or insome cases, oligonucleotides, which bind to human stanniocalcin-alphaand eliminate its function. Antagonists also include polypeptides whichbind to human stanniocalcin-alpha receptors and effectively block thereceptor from human stanniocalcin-alpha. These polypeptides are proteinswhich are closely related to human stanniocalcin-alpha but have lostnatural biological function, an example is a mutated form of humanstanniocalcin-alpha.

Human stanniocalcin-alpha antagonists also include anti-senseconstructs. Antisense technology can be used to control gene expressionthrough triple-helix formation or antisense DNA or RNA, both of whichmethods are based on binding of a polynucleotide to DNA or RNA. Forexample, the 5′ coding portion of the polynucleotide sequence, whichencodes for the mature polypeptides of the present invention, is used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple helix—see Leeet al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456(1988); and Dervan et al., Science, 251: 1360 (1991)), therebypreventing transcription and the production of humanstanniocalcin-alpha. The antisense RNA oligonucleotide hybridizes to themRNA in vivo and blocks translation of the mRNA molecule into the humanstanniocalcin-alpha polypeptide (Antisense—Okano, J. Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of humanstanniocalcin-alpha protein.

Human stanniocalcin-alpha antagonists also include small molecules whichbind to the active site of the polypeptide making it unable to impartbiological function. Examples of small molecules include but are notlimited to small peptides or peptide-like molecules.

The antagonists may be employed to block the stimulation of boneresorption by a high concentration of human stanniocalcin-alpha and,accordingly, may be employed to treat osteoporosis.

The human stanniocalcin-alpha antagonists may also be employed to treathypocalcemia and Paget's disease among other disorders where an increasein calcium levels is desired. The antagonists may be employed in acomposition with a pharmaceutically acceptable carrier, e.g., ashereinafter described.

The human stanniocalcin-alpha polypeptides of the present invention, andagonist and antagonist compounds, may be employed in combination with asuitable pharmaceutical carrier. Such pharmaceutical compositionscomprise a therapeutically effective amount of the polypeptide, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepharmaceutical compositions may be employed in conjunction with othertherapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, the pharmaceutical compositions will be administered in anamount of at least about 10 g/kg body weight and in most cases they willbe administered in an amount not in excess of about 8 mg/Kg body weightper day. In most cases, the dosage is from about 10 g/kg to about 1mg/kg body weight daily, taking into account the routes ofadministration, symptoms, etc.

The human stanniocalcin-alpha polypeptides, and agonists and antagonistswhich are also polypeptides, may be employed in accordance with thepresent invention by expression of such polypeptides in vivo, which isoften referred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, and-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the -actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, -2,-AM, PA12, T19-14X, VT-19-17-H2, CRE, CRIP, GP+E-86, GP+envAm12, and DANcell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14(1990), which is incorporated herein by reference in its entirety. Thevector may transduce the packaging cells through any means known in theart. Such means include, but are not limited to, electroporation, theuse of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of the stanniocalcin-alphagene as part of a diagnostic assay for detecting diseases orsusceptibility to diseases related to the presence of mutated humanstanniocalcin-alpha. Such diseases are related to an under-expression ofhuman stanniocalcin-alpha, for example, hypertension.

Individuals carrying mutations in the human stanniocalcin-alpha gene maybe detected at the DNA level by a variety of techniques. Nucleic acidsfor diagnosis may be obtained from a patient's cells, such as fromblood, urine, saliva, tissue biopsy and autopsy material. The genomicDNA may be used directly for detection or may be amplified enzymaticallyby using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior toanalysis. RNA or cDNA may also be used for the same purpose. As anexample, PCR primers complementary to the nucleic acid encoding humanstanniocalcin-alpha can be used to identify and analyze humanstanniocalcin-alpha mutations. For example, deletions and insertions canbe detected by a change in size of the amplified product in comparisonto the normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabeled human stanniocalcin-alpha RNA oralternatively, radiolabeled human stanniocalcin-alpha antisense DNAsequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamidine gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region of the sequence is used to rapidly select primersthat do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers are then used forPCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 50 or 60bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37 C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1 Bacterial Expression and Purification of HumanStanniocalcin-Alpha

The DNA sequence encoding human stanniocalcin-alpha ATCC™# 75831 (SEQ IDNO:11), is initially amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the stanniocalcin-alphacoding sequences. The 5′ oligonucleotide primer has the sequence 5′GACTACATGTGTGCCGAGCGGCTGGG 3′ (SEQ ID NO:3) contains a Afl IIIrestriction enzyme site and 20 nucleotides of stanniocalcin-alpha codingsequence starting from the presumed methionine start codon. The 3′sequence 5′ GACTAGATCTCTCCTGGGCTCTGGGAGGTG 3′ (SEQ ID NO:4) containscomplementary sequences to a Bgl II site and is followed by 20nucleotides of stanniocalcin-alpha. A pQE-60 vector (Qiagen, Inc. 9259Eton Avenue, Chatsworth, Calif., 91311) encodes antibiotic resistance(Amp^(r)), a bacterial origin of replication (ori), an IPTG-regulatablepromoter operator (P/O), a ribosome-binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-60 is digested with Afl III and Bgl II.The amplified sequences are ligated into pQE-60 after digestion with AflIII and Bgl II and are inserted in frame with the sequence encoding forthe histidine tag and the RBS. The ligation mixture is then used totransform the E. coli strain M15/rep 4 available from Qiagen by theprocedure described in Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989). M15/rep4contains multiple copies of the plasmid pREP4, which expresses the lacIrepressor and also confers kanamycin resistance (Kan^(r)). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis. Clones containing thedesired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 μg/ml) and Kan (25 μg/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.₆₀₀) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation (20 mins at 6000 Xg). The cell pellet issolubilized in the chaotropic agent 6 Molar Guanidine HCl. Afterclarification, solubilized stanniocalcin-alpha is purified from thissolution by chromatography on a Nickel-Chelate column under conditionsthat allow for tight binding by proteins containing the 6-His tag(Hochuli, E. et al., Genetic Engineering, Principles & Methods, 12:87-98(1990)). Protein renaturation out of GnHCl can be accomplished byseveral protocols (Jaenicke, R. and Rudolph, R., Protein Structure—APractical Approach, IRL Press, New York (1990)). Initially, stepdialysis is utilized to remove the GnHCL. Alternatively, the purifiedprotein isolated from the Ni-chelate column can be bound to a secondcolumn over which a decreasing linear GnHCL gradient is run. The proteinis allowed to renature while bound to the column and is subsequentlyeluted with a buffer containing 250 mM Imidazole, 150 mM NaCl, 25 mMTris-HCl pH 7.5 and 10% Glycerol. Finally, soluble protein is dialyzedagainst a storage buffer containing 5 mM Ammonium Bicarbonate. Thepurified protein was analyzed by SDS-PAGE.

EXAMPLE 2 Expression of Recombinant Human Stanniocalcin-Alpha in COSCells

The expression of plasmid, stanniocalcin-alpha HA is derived from avector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin ofreplication, 2) ampicillin resistance gene, 3) E. coli replicationorigin, 4) CMV promoter followed by a polylinker region, an SV40 intronand polyadenylation site. A DNA fragment encoding the entirestanniocalcin-alpha precursor and a HA tag fused in frame to its 3′ endwas cloned into the polylinker region of the vector, therefore, therecombinant protein expression is directed under the CMV promoter. TheHA tag correspond to an epitope derived from the influenza hemagglutininprotein as previously described (I. Wilson, H. Niman, R. Heighten, ACherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusionof HA tag to the target protein allows easy detection of the recombinantprotein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding stanniocalcin-alpha was constructed by PCR onthe original express sequence tag (EST) cloned using two primers: the 5′primer 5′ GAC TAAGCTTATGTGTGCCGAGCGGCTGGGC 3′ (SEQ ID NO:5) contains aHind III site followed by 21 nucleotides of stanniocalcin-alpha codingsequence starting from the initiation codon; the 3′ sequence 5′GACTTCTAGACTAAGCGTAGTCTGGGACGTCG TATGGGTACTCCTGGGCTCTGGGAGGTG 3′ (SEQ IDNO:6) contains complementary sequences to an Xba I site, translationstop codon, HA tag and the last 20 nucleotides of thestanniocalcin-alpha coding sequence (not including the stop codon).Therefore, the PCR product contains a Hind III site, stanniocalcin-alphacoding sequence followed by HA tag fused in frame, a translationtermination stop codon next to the HA tag, and an Xba I site. The PCRamplified DNA fragment and the vector, pcDNAI/Amp, were digested withHind III and Xba I restriction enzyme and ligated. The ligation mixturewas transformed into E. coli strain SURE (available from StratageneCloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037)the transformed culture was plated on ampicillin media plates andresistant colonies were selected. Plasmid DNA was isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment. For expression of the recombinantstanniocalcin-alpha, COS cells were transfected with the expressionvector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989)). The expression of the stanniocalcin-alpha HA protein wasdetected by the radiolabelling and immunoprecipitation method (E.Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, (1988)). Cells were labeled for 8 hours with³⁵S-cysteine two days post transfection. Culture media was thencollected and cells were lysed with detergent (RIPA buffer (150 mM NaCl,1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I.et al., Id. 37:767 (1984)). Both cell lysate and culture media wereprecipitated with an HA specific monoclonal antibody. Proteinsprecipitated were analyzed on 15% SDS-PAGE gels.

EXAMPLE 3 Cloning and Expression of Human Stanniocalcin-Alpha Using theBaculovirus Expression System

The DNA sequence encoding the full-length stanniocalcin-alpha protein,ATCC™# 75831 (SEQ ID NO: 11), is amplified using PCR oligonucleotideprimers corresponding to the 5′ and 3′ sequences of the gene:

The 5′ primer has the sequence 5′ GACTGGATCCGCCACCATGTGTGCCG AGCGGCTGGGC3′ (SEQ ID NO:7) and contains a BamHI restriction enzyme site (in bold)followed by 6 nucleotides resembling an efficient signal for theinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.,196:947-950 (1987) which is just behind the first 21 nucleotides of thestanniocalcin-alpha gene (the initiation codon for translation “ATG” isunderlined).

The 3′ primer has the sequence 5′ GACTGGTACCCTACTCCTGGGCT CTGGG AGG 3′(SEQ ID NO:8) and contains the cleavage site for the restrictionendonuclease Asp 718 and 21 nucleotides complementary to the 3′ sequenceof the stanniocalcin-alpha gene. The amplified sequences are isolatedfrom a 1% agarose gel using a commercially available kit (“Geneclean™,”BIO 101 Inc., La Jolla, Calif.). The fragment is then digested with theendonucleases BamHI and Asp 718 and then purified again on a 1% agarosegel. This fragment is designated F2.

The vector pRG1 (modification of pVL941 vector, discussed below) is usedfor the expression of the stanniocalcin-alpha protein using thebaculovirus expression system (for review see: Summers, M. D. and Smith,G. E. 1987, A manual of methods for baculovirus vectors and insect cellculture procedures, Texas Agricultural Experimental Station Bulletin No.1555). This expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby the recognition sites for the restriction endonucleases BamHI and Asp718. The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of cotransfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid is digested with the restriction enzymes BamiHI and Asp 718and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The DNA is then isolated from a 1% agarosegel using the commercially available kit (“Geneclean™” BIO 101 Inc., LaJolla, Calif.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNAligase. E.coli HB101 cells are then transformed and bacteria identifiedthat contained the plasmid (pBac-stanniocalcin-alpha) with thestanniocalcin-alpha gene using the enzymes BamHi and Asp 718. Thesequence of the cloned fragment is confirmed by DNA sequencing.

5 μg of the plasmid pBac-stanniocalcin-alpha is cotransfected with 1.0μg of a commercially available linearized baculovirus (“BaculoGoldTmbaculovirus DNA”, Pharmingen, San Diego, Calif.) using the lipofectionmethod (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmidpBac-stanniocalcin-alpha are mixed in a sterile well of a microtiterplate containing 50 μl of serum free Grace's medium (Life TechnologiesInc., Gaithersburg, Md.). Afterwards 10 ∥l Lipofectin™ plus 90 μlGrace's medium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added dropwise to the Sf9insect cells (ATCC™ CRL 1711) seeded in a 35 mm tissue culture platewith 1 ml Grace's medium without serum. The plate is rocked back andforth to mix the newly added solution. The plate is then incubated for 5hours at 27° C. After 5 hours the transfection solution is removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. The plate is put back into an incubator andcultivation continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the viruses are added to the cells,blue stained plaques are picked with the tip of an Eppendorf pipette.The agar containing the recombinant viruses is then resuspended in anEppendorf tube containing 200 μl of Grace's medium. The agar is removedby a brief centrifugation and the supernatant containing the recombinantbaculoviruses is used to infect Sf9 cells seeded in 35 mm dishes. Fourdays later the supernatants of these culture dishes are harvested andthen stored at 4° C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-stanniocalcin-alpha at a multiplicity of infection (MOI)of 2. Six hours later the medium is removed and replaced with SF900 IImedium minus methionine and cysteine (Life Technologies Inc.,Gaithersburg). 42 hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵Scysteine (Amersham) are added. The cells are further incubated for 16hours before they are harvested by centrifugation and the labeledproteins visualized by SDS-PAGE and autoradiography (FIG. 5). In FIG. 5the gel indicates that stanniocalcin-alpha exists as a homodimer.

EXAMPLE 4 Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer containing an EcoRI site and the 3′ primerfurther includes a HindeIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1. An isolated polynucleotide comprising a member selected from thegroup consisting of: (a) a polynucleotide encoding a mature polypeptidehaving the deduced amino acid sequence of SEQ ID NO:2 or a fragment,analog or derivative of said polypeptide; (b) a polynucleotide encodinga mature polypeptide having the amino acid sequence encoded by the cDNAcontained in ATCC Deposit No. 75831 or a fragment, analog or derivativeof said polypeptide.
 2. The polynucleotide of claim 1 wherein thepolynucleotide is RNA.
 3. The polynucleotide of claim 1 wherein thepolynucleotide is genomic DNA.
 4. The polynucleotide of claim 1 whereinsaid polynucleotide encodes a mature polypeptide having the deducedamino acid sequence of SEQ ID NO:2.
 5. The polynucleotide of claim 1wherein said polynucleotide encodes a mature polypeptide encoded by thecDNA of ATCC Deposit No.
 75831. 6. The polynucleotide of claim 1comprising the DNA sequence of Sequence ID No. 1 encoding the maturepolypeptide of Sequence ID No.
 2. 7. A vector containing the DNA ofclaim
 2. 8. A host cell genetically engineered with the vector of claim7.
 9. A process for producing a polypeptide comprising: expressing fromthe host cell of claim 8 the polypeptide encoded by said DNA.
 10. Aprocess for producing cells capable of expressing a polypeptidecomprising genetically engineering cells with the vector of claim
 7. 11.An isolated polynucleotide hybridizable to the polynucleotide of claim 1and having at least a 70% identity thereto.
 12. A polypeptide comprisinga member selected from the group consisting of (i) a mature polypeptidehaving the deduced amino acid sequence of Sequence ID No. 2 andfragments, analogs and derivatives thereof; and (ii) a maturepolypeptide encoded by the cDNA of ATCC Deposit No. 75831 and fragments,analogs and derivatives of said polypeptide.
 13. An antibody against thepolypeptide of claim
 12. 14. An antagonist against the polypeptide ofclaim
 12. 15. An agonist to the polypeptide of claim
 12. 16. A methodfor the treatment of a patient having need of human stanniocalcin-alphacomprising: administering to the patient a therapeutically effectiveamount of the polypeptide of claim
 12. 17. A method for the treatment ofa patient having need to inhibit stanniocalcin-alpha comprising:administering to the patient a therapeutically effective amount of theantagonist of claim
 15. 18. The method of claim 16 wherein saidtherapeutically effective amount of the polypeptide is administered byproviding to the patient DNA encoding said polypeptide and expressingsaid polypeptide in vivo.
 19. A process for identifying agonists orantagonists comprising: combining a mammalian cell which expresses astanniocalcin-alpha receptor on the surface thereof, labeled calcium andthe compound to be screened and further in the presence ofstanniocalcin-alpha when the screening is for an antagonist; anddetermining whether the compound stimulates or inhibits calcium uptake.20. A process for diagnosing a disease or a susceptibility to a diseaserelated to an under-expression of human stanniocalcin-alpha polypeptidethe polypeptide of claim 12 in a host comprising: determining in asample derived from a host a mutation in the human stanniocalcin-alphanucleic acid sequence encoding the mature polypeptide of Sequence ID No.2.